Circulating blood cells are constantly replaced by newly developed cells. Replacement blood cells are formed in a process termed hematopoiesis which involves the production of at least eight mature blood cell types within two major lineages: (1) myeloid which includes red blood cells (erythrocytes), macrophages (monocytes), eosinophilic granulocytes, megakaryocytes (platelets), neutrophilic granulocytes, basophilic granulocytes (mast cells); and (2) lymphoid which includes T lymphocytes, and B lymphocytes, Burgess and Nicola, Growth Factors and Stem Cells (Academic Press, New York, 1983). Much of the control of blood cell formation is mediated by a group of interacting glycoproteins termed colony stimulating factors (CSFs). These glycoproteins are so named because of the in vivo and in vitro assays used to detect their pressence. Techniques for the clonal culture of hematopoietic cells in semisolid culture medium have been especially important in the development of in vitro assays. In such cultures, individual progenitor cells (i.e., cells developmentally committed to a particular lineage, but still capable of proliferation) are able to proliferate to form a colony of maturing progeny in a manner which is believed to be essentially identical to the comparable process in vivo. The role of CSFs in hematopoiesis is the subject of many recent reviews, e.g. Metcalf, The Hemopoietic Colony Stimulating Factors (Elsevier, New York, 1984); Metcalf, Science, Vol. 229, pgs. 16-22 (1985); Nicola et al., Immunology Today, Vol. 5, pgs. 76-80 (1984); Whetton et al., TIBS, Vol. 11, pgs. 207-211 (1986), Clark and Kamen, Science, Vol. 236, pgs. 1229-1237 ( 1987) and Sachs, Science, Vol. 238, pgs. 1374-1379 (1987).
The detection, isolation and purification of these factors is extremely difficult, being frequently complicated by the complexity of the biological fluids they are typically located in, and their very low concentrations. As more CSFs become available, primarily through molecular cloning, interest has heightened in finding clinical applications for them. Because of physiological similarities to hormones (e.g., soluble factors, growth mediators, action via cell receptors), potential uses of CSFs have been analogized to the current uses of hormones, e.g. Dexter, Nature, Vol. 321, pg. 198 (1986). Their use has been suggested for several clinical situations where the stimulation of blood cell generation would be desirable, such as for rehabilitative therapy after chemotherapy or radiation therapy of tumors, treatment of myeloid hypoplasias, treatment of neutrophil deficiency, treatment to enhance hematopoietic regeneration following bone marrow transplantation, and treatment to increase host resistance to established infections, e.g. Dexter (cited above), Metcalf, Science (cited above), and Clark and Kamen (cited above). Recently, recombinant human GM-CSF has been shown to produce a dose-dependent increases in circulating leukocyte count in severely leukopenic AIDS patients, Groopman, Cell, Vol. 50, pgs. 5-6 (1987).
CSFs are also believed to play a role in the development and progression of myeloid leukemias. Myeloid leukemias are clonal neoplasms of granulocyte-macrophage precursor cells, which fall into two major groups--chronic myeloid leukemia (CML) and acute myeloid leukemia (AML). CML is characterized by expansion in the marrow of the granulocyte-monocyte population at all stages of maturation with massive enlargement of hematopoietic populations in the spleen and blood. While chemotherapy is successful in reducing the excessive size of the leukemic cell populations conventional regimens have not succeeded in preventing terminal acute transformation (of progressively higher proportions of cells into immature or abnormal forms) or in extending the life spans of afflicted patients, Metcalf (cited above, 1984).
AML is characterized by an accumulation of immature granulocyte-monocyte blast cells with often little or no evidence of maturing granulocyte-monocyte cells. The disease primarily involves the bone marrow, and spleen enlargement usually is only moderate. Total blood nucleated cells may or may not be elevated but there is a high proportion of immature blast cells associated with relatively few mature cells. There is usually an associated anemia, thrombocytopenia and a relative absence in the marrow and peripheral blood of mature granulocytes and monocytes. Death usually results from uncontrollable hemorrhage or overwhelming infections, Metcalf (cited above, 1984).
It is believed that both forms of leukemia are driven by abnormal production of, or responsiveness to, colony stimulating factors, particularly GM-CSF. In particular, it has been shown that leukemic cells from some AML patients are capable of autonomous in vitro proliferation because they express GM-CSF constitutively, and that such autonomous proliferation can be inhibited by the addition of GM-CSF neutralizing antiserum, Young et al., Blood, Vol. 68, pgs. 1178-1181 (1986).
It is believed that myeloid leukemias, in particular AML, may be treated by blocking the ability of GM-CSF to stimulate cell growth. Blocking agents can be derived from monoclonal antibodies specific for human GM-CSF.
Such monoclonal antibodies have other uses, including detection, measurement, and purification of GM-CSF. An important aspect of any therapy involving drugs is the ability to predict and/or monitor concentration levels in the blood or other patient body fluids. Monoclonal antibodies are widely used for this purpose, e.g. Springer, ed., Hybridoma Technology in the Biosciences and Medicine (Plenum Press, N.Y., 1985); and U.S. Pat. Nos. 4,562,003; 4,486,530; and 4,255,329.
In the production of genetically engineered proteins such as human GM-CSF, separation of the expressed protein from the transformed host cells and/or their culture supernatants is a major problem. Frequently separation procedures involve one or more passes of crude material through immunoadsorbent columns. Monoclonal antibodies specific for the protein to be purified are crucial elements of such columns. Such monoclonal antibodies can also be used to measure the degree of purification achieved by a particular protocol, e.g. by "Western" blot analysis, Burnette, Anal. Biochem., Vol. 112, pgs. 195-203 (1981).
From the foregoing, it is evident that the availability monoclonal antibodies specific for human GM-CSF could facilitate the medical and veterinary applications of GM-CSF by providing alternative methods to bioactivity measurements for its detection, purification, and measurement.